MUTATIONS IN THE THIOREDOXIN SITES OF PROTEIN DISULFIDE-ISOMERASE REVEAL FUNCTIONAL NONEQUIVALENCE OF THE N-TERMINAL AND C-TERMINAL DOMAINS

Protein disulfide isomerase (PDI), a foldase of the endoplasmic rectic ulum, is a multifunctional protein that catalyzes the formation and is omerization of disulfide bonds during protein folding. The wild-type p rotein contains two redox active thiol/disulfide sites near the N and C terminus that are homologous to the redox center of thioredoxin. Usi ng site-directed mutagenesis, both cysteines of each of the thioredoxi n-like centers, (C35S,C38S) and (C379S,C382S) were replaced by serines . In addition, a mutant PDI was constructed with all four of the activ e cysteines mutated to serine (C35S,C38S,C379S,C382S). The activity of the wild-type and mutant proteins in the oxidative renaturation of re duced, denatured RNase was analyzed over a wide range of RNase concent rations, PDI concentrations, and glutathione redox buffers composition s. All mutants, including the construct with no functional thioredoxin centers, have measurable disulfide isomerase activity. Both of the th ioredoxin-like sites contribute some to apparent steady-state binding (K-m) and catalysis at saturating substrate concentrations (k(cat)); h owever their contributions are not equivalent. At saturating concentra tions of RNase, the mutant with an inactivated C-terminal active site (k(cat) = 0.72 +/- 0.06 min(-1)) retains near wild-type activity (k(ca t) = 0.76 +/- 0.02 min(-1)), while the N-terminal mutant exhibits a si gnificantly lower k(cat) (0.24 +/- 0.01 min(-1)). The K-m for RNase is elevated for the C-terminal mutant (K-m = 29 +/- 4 mu M) while the N- terminal mutant (K-m = 7.1 +/- 1.1 mu M) exhibits a wild-type K-m (6.9 +/- 0.8 mu M). The larger K-m for the C-terminal mutant (4.2 times wi ld-type) and the lower k(cat) of N-terminal mutant (32% of wild-type) suggest that the C-terminal region contributes more to apparent steady -state substrate binding, and the N-terminal region contributes more t o catalysis at saturating concentrations of substrate. Despite their c omplementary roles in catalysis, the thioredoxin-like centers exhibit the same dependence on the glutathione redox buffer composition as evi denced by the equivalent K-ox values for the wild-type (47 +/- 1 mu M) , N-terminal mutant (43 +/- 3 mu M), and C-terminal mutant (44 +/- 1 m u M). The mutant with both thioredoxin sites mutated displays a low bu t detectable level of disulfide-isomerase activity (0.5% of wild-type) that can be observed at high PDI concentrations. At high RNase concen trations (greater than or equal to 26 mu M), wild-type PDI and all of the mutants catalyze intermolecular RNase aggregation in a nucleation growth reaction that is first order in PDI but fourth order with respe ct to RNase. Disulfide formation is required for RNase aggregation. Th is behavior is similar to the anti-chaperone activity previously obser ved for wild-type PDI (Puig, A., and Gilbert, H. F. (1994) J. Biol Che m. 269, 7764-7771; Puig, A., Lyles, M. M., Noiva, R., and Gilbert, H. F. (1994) J. Biol. Chem. 269, 19128-19135).